A fundamental question regarding receptor-G protein interaction is whether different agonists can lead a receptor to different intracellular signaling pathways. Our previous studies have demonstrated that while most beta2-AR agonists activate both Gs and Gi proteins, fenoterol, a full agonist of beta2-AR, selectively activates Gs protein. Fenoterol contains two chiral centers and may exist as four stereoisomers. We have synthesized a series of stereoisomers of fenoterol and its derivatives and characterized their receptor binding and pharmacological properties. We tested the hypothesis that the stereochemistry of an agonist determines selectivity of receptor coupling to different G protein(s). We found that the R,R-isomers of fenoterol and methoxyfenoterol exhibited more potent effects to increase cardiomyocyte contraction than their S,R-isomers. Importantly, while R,R-fenoterol and R,R-methoxyfenoterol preferentially activate Gs signaling, their S,R-isomers were able to activate both Gs and Gi proteins as evidenced by the robust pertussis toxin-sensitivities of their effects on cardiomyocyte contraction and on phosphorylation of extracellular signal-regulated kinase 1/2. The differential G protein selectivities of the fenoterol stereoisomers were further confirmed by photoaffinity labeling studies on Gs, Gi2 and Gi3 proteins. The inefficient Gi signaling with the R,R-isomers is not caused by the inability of the R,R-isomers to trigger the PKA-mediated phosphorylation of the beta2-AR, since the R,R-isomers also markedly increased phosphorylation of the receptor at serine262 by PKA. We conclude that in addition to receptor subtype and phosphorylation status, the stereochemistry of a given agonist plays an important role in determining receptor-G protein selectivity and downstream signaling events.